PROJECT SUMMARY Through cohesive advancements in protein engineering, bioconjugation chemistry and sophisticated nanotechnology, innovations in targeted nanotherapeutics have made significant progress in increasing the specificity of anticancer nanomedicines. However, as a consequence of the complexity of modular targeted nanoconjugates (TNCs), in vivo selectivity, and subsequent treatment outcomes, frequently suffer setbacks. The capacity to accurately resolve intratumoral molecular and cellular selectivity of TNCs in vivo is oftentimes thwarted by dominant macrophysiological factors. Such factors include macro-scale quantification of TNC tumor delivery, which provides little insight into micro-scale cancer cell-specific molecular binding events and internalization of TNCs present within the interstitium. Additional factors include variable pharmacokinetics of non-selective nanoconjugate controls, and unparalleled physiologies of target-null tumors; both which hamper the accurate experimental comparisons needed to evaluate novel TNCs. To this end, Dual-Tracer Fluorescence Imaging (DT-FI) will be deployed, which provides accurate in vivo quantitation of tumor target molecular binding of TNCs via a Binding Potential metric, a combined measure of binding affinity and respective tumor target receptor concentration. Head and neck squamous cell carcinoma (HNSCC) is debilitating and manifests at critical anatomical sites, requiring highly selective treatments to preserve function and aesthetics. Aside from indiscriminate surgery, chemo- and radiotherapy, confined spatiotemporal control of cytotoxicity of HNSCC can be imparted by photodynamic therapy (PDT), a promising modality used to manage cancers by the light-activation of photosensitizer (PS) agents. In an effort to improve the cellular selectivity of HNSCC therapy, PS-embedded, chemo-loaded liposomes will be employed in this proposal as a model nanoplatform to surface-graft engineered recombinant targeting moieties. DT-FI will be leveraged as a critical means of directing the engineering of the photoactive TNCs to improve outcomes of PDT-based treatments in HNSCC. To guide Dr. Obaid's transition to independence, a mentoring committee has been assembled to complement his training in chemical nanoscience. Mentorship by Dr. Tayyaba Hasan will train Dr. Obaid on PDT-based cancer nanomedicines and tumor biology response. Co-mentorship by Dr. Brian Pogue will train Dr. Obaid in quantitative in vivo DT-FI. Additional distinguished members include Dr. Brian Seed, a specialist in protein engineering, Dr. Andrew Tsourkas, a nanoconjugation expert, Dr. Eben Rosenthal, Otolaryngologist and pioneer in HNSCC imaging, and Dr. William Faquin, a HNSCC pathophysiology expert. As a well-defined and distinct transition to independence, the infrastructure established in the mentored phase will be adapted to the engineering of multi-specific photo-chemotherapeutic nanoplatforms tailored for in vivo heterogeneity of HNSCC models. The approach established in this K99/R00 mechanism will enable future designs of various TNCs, as directed by in vivo DT-FI, with the capacity for targeting dynamic disease-specific molecular targets.